E. coli thioredoxin is a highly efficient catalyst for both the reduction of disulfides as well as the oxidation of dithiols for both protein and small molecule substrates. Previous research from other laboratories have used NMR chemical shift titration to determine the pK values for the two active site cysteines Cys 32 and Cys 35 as well as for the nearby buried Asp 26. Due to the marked interaction between these sites interpretation of this data has been controversial. Although these previous experiments have been carried out on the reduced form of the protein which is germane to the initial attack of Cys 32 on a target disulfide, to understand the nucleophilicity of the buried Cys 35 thiol pH titrations are needed for a model of the mixed disulfide intermediate. These titrations have demonstrated the marked dependence of the Cys 35 resonances on the Asp 26 titration (rather than due to the titration of Cys 32 as earlier interpreted). The pK of Cys 35 in this derivative differs appreciably from earlier estimates. Standard enzyme kinetics studies have indicated that the rate of reduction of the enzyme by small molecule dithiols is faster than 1 s-1 at pH 7. By poising the redox potential of the solution to match that of E. coli thioredoxin, magnetization transfer experiments are being carried out so as to determine the unidirectional rate constants for the redox transition.